Mastoparan, a peptide toxin from wasp venom, causes the activation of GTP- binding regulatory proteins (G proteins) by promoting GTP binding in a manner strikingly similar to that of cell surface receptors. mastoparan (MP), a cationic, amphiphilic helix, is also structurally similar to putative G protein-binding domains on these receptors. I propose to study the mechanism and structural basis of the G protein activation by MP and related amphiphilic compounds as a model for receptor-G protein coupling and to develop G protein-specific regulatory peptides as probes of cellular regulation. (1) We will develop MP analogs that are more potent and more selective G protein activators and inhibitors. Structure-activity analysis will use kinetic assays with purified G proteins and recombinant alpha subunits, physical studies of MP-G protein complexes, and computer assisted modeling. (2) Affinity cross-linking between MP and G proteins will be performed to determine the MP-binding site, presumably the receptor binding-site as well. We will evaluate competition between receptor and MP for binding to G proteins to evaluate the identity of their binding sites. (3) Circular dichroism, 19F-NMR, and fluorescence spectroscopy will be used to study the structural basis of MP-G protein binding. Two-dimensional transferred NOE of 1H-NMR (500 MHz) will be used to determine the conformation of MP when bound to G proteins. The conformational basis of MP binding to G proteins will be compared with its binding to calmodulin, which binds many amphiphilic peptides. (4) We will study the mechanism by which MP and related compounds activate G proteins or block activation using kinetic and ligand binding assays. We will clarify the roles of the G protein betagamma subunits and Mg2+ on MP action. We will extend these studies to small GTP-binding proteins, particularly p21ras, which are not known to be regulated by receptors. (5) MP analogs will be used to study G protein-mediated signaling in cells. Affinity crosslinking of MP targets in cells, measurements of MP uptake and its mechanism and the use of MP as an affinity chromatographic ligand will be included in these experiments.